Enteric bacteria such as Escherichia coli are a major cause of human disease. These bacteria produce curli, extracellular protein fibers that contribute to virulence. In addition to being important pathogenicity factors, with roles in host colonization, immune activation, and cell invasion, curli act as the major proteinaceous scaffold for bacterial biofilms. Curli are biophysically classified as an amyloid fiber because they adopt a cross -strand fibrillar structure common to all amyloids. Amyloids have historically been associated with protein misfolding and cellular toxicity, especially neurotoxicity. Curli are not te products of protein misfolding, but instead are the result of an evolved biogenesis pathway. It is now clear that functional amyloids are widespread, with examples found in nearly all facets of cellular life. The curli system in E. coli provides a rich genetic and biochemical toolbox for the study of amyloid formation. Our long-term goal is to understand how E. coli builds an amyloid fiber, so that new therapies can be developed that rationally target this critical biological process. Knowledge gained here will have implications for both microbial pathogenesis and protein folding and misfolding. Our previous discoveries have contributed to a curli assembly model where the main fiber component CsgA and the minor subunit CsgB are secreted through the outer membrane via the lipoprotein CsgG. CsgB attaches to the surface of the cell and templates the folding of CsgA into an amyloid fiber. CsgE, an accessory protein with chaperone-like activity against CsgA, is also required for curli subunit secretion. In order to rationally develop therapeutics against virulence factors such as curli, we must better understand curli biogenesis and function. In Aim 1 we will focus on further developing and testing the curli biogenesis model. The roles of the chaperonelike accessory protein CsgE and the outer membrane lipoprotein CsgG in directing efficient CsgA transport through the periplasm will be explored. We will also investigate the mechanics of a newly discovered periplasmic chaperone activity that is dependent on the CsgC protein. In Aim 2 we will assess the ability of previously constructed CsgA and CsgB mutants to support biological function in well-developed in vivo biofilm assays. Furthermore, the specificity of amyloid seeding will be tested in polymicrobial biofilms. Finally, in Aim 3 we will develop small molecules with amyloid-altering capabilities. In collaboration with Fredrik Almqivst at Ume University in Sweden, we have already identified molecules that discourage CsgA polymerization. We will further characterize these 2-pryidinone variants and screen a second generation of compounds for antiamyloid properties. The curli system has evolved as an amyloid on purpose, and we will exploit this system in order to better understand global tenets of amyloid formation, microbial pathogenesis, and biofilm biology.